Engineered autologous adult-derived stem cells represent a unique therapeutic approach to deliver localized tumoricidal factors to inhibit cancer growth and proliferation. While in its technological infancy, bioengineered adult-derived mesenchymal stem cells (MSCs) show early promise as a delivery system of therapeutic cytokines in a variety of clinical applications. With the ease of clinical isolation of autologous MSCs and their demonstrated robust tropism for a variety of tumors in vitro and in vivo, engineered MSCs have been proposed as therapeutic anticancer vehicles. Breast cancer represents a broad-spectrum cancer with diverse morphological and molecular features and varied clinical outcomes. Clinical experience has shown that a multi-target approach to breast cancer therapy is usually superior to single agent treatments. Our long-range goal is to actualize a tractable, versatile, synthetic chromosome-based platform (termed ACE chromosome), onto which multiple therapeutics may be placed for directing a multi-targeted approach to breast cancer therapy. The ACE chromosome, an autonomous chromosome-based circuit board designed to contain approximately 70 site-specific, recombination acceptor sites that can carry single or multiple copies of genes of interest, is amenable to MSC bioengineering. Pursuant of our long-range goal, we propose to bioengineer MSCs with an ACE chromosome in order to express, on demand, two anti-tumor factors-interferon beta (IFNb) and the EGFR/MET receptor antagonist decorin (DCN). The central hypothesis of this application is that the ACE synthetic chromosome, simultaneously expressing these two antitumor factors from MSCs will efficiently inhibit growth and promote death of breast tumor cells as compared to either factor alone. The experiments outlined in this proposal will serve as proof of concept that the ACE system provides a novel cytoreagent, bioengineering strategy applicable to cancer therapeutics. We plan to test our hypothesis and accomplish the objective of this application by pursuing the following specific aims: 1) Bioengineer murine MSCs to carry an ACE chromosome and express, on demand, IFNb and DCN and demonstrate their ability to inhibit cancer cell growth in vitro and reduce tumor burden in an immunocompetent, syngeneic murine model of breast cancer, and 2) Determine if the proliferation human triple negative breast cancer cell lines can be inhibited in vitro by the production of IFNb and DCN from ACE engineered human MSCs. The proposed preclinical proof of concept described in this application is innovative as it conjoins the ACE-chromosome bioengineering technology with MSCs thus potentially providing a powerful and novel cytoreagent for delivering sustained expression of a multiplicity of genes targeted towards breast cancer cells. The ACE synthetic chromosome technology provides a means by which multiple therapeutics can leverage multi-targeted and, potentially, synergistic mechanisms thereby producing enhanced therapeutic efficacy in cancers that are often refractory to current therapies.